Fluorescent dyes are widely used in biological research and medical diagnostics. Fluorescent dyes are superior to conventional radioactive materials because fluorescent dyes are typically sufficiently sensitive to be detected, less expensive and less toxic. In particular, a diversity of fluorophores with a distinguishable color range has made it more practical to perform multiplexed assays capable of detecting multiple biological targets in parallel. The ability to visualize multiple targets in parallel is often required for delineating the spatial and temporal relationships amongst different biological targets in vitro and in vivo. In addition, the generation of a wide range of fluorescent dyes has opened a new avenue for conducting high-throughput and automated assays, thus dramatically reducing the unit cost per assay. Moreover, the low toxicity of fluorescent dyes provides ease of handling in vitro, and also renders it safer for imaging biological activities in vivo.
Despite the various advantages of fluorescent dyes, conventional dyes have a number of profound limitations. For example, conventional fluorescent dyes are typically prone to inter-dye quenching, a phenomenon known to diminish the effective brightness of the dyes. It is a common practice to conjugate a given target with multiple dye molecules in order to maximize the brightness of the labeled target, e.g., a biomolecule such as protein or DNA. For many conventional fluorescent dyes, the fluorescence intensity of the labeled target is often not directly proportional to the number of attached dye molecules, but rather less than the predicted intensity due to, e.g., quenching amongst the multiple dyes attached to the target. Such quenching effect can be attributed to, in part, the physical interaction amongst the attached dye molecules, which may lead to formation of nonfluorescent dye dimers. Dimer formation may be driven by hydrophobic interaction. Because many traditional fluorescent dyes, such as various rhodamine dyes and cyanine dyes, are highly hydrophobic aromatic compounds, these commonly used dyes are particularly prone to forming dimers on labeled biomolecules. Adding sulfonate groups to a dye has been shown to reduce dimer formation. See, e.g., U.S. Pat. Nos. 5,268,486 and 6,977,305, 6,130,101 and Panchuk-Voloshina, et al. J. Histochem. Cytochem. 47(9), 1179 (1999). However, while sulfonation may reduce dimer formation, it also introduces negative charges into a biomolecule, and thus may increase the risk of disrupting the biological activity of the labeled biomolecule. Furthermore, dyes substituted with sulfonates alone may exhibit a shorter serum half-life when used in vivo in a subject.
Another limiting factor for conventional fluorescent dyes is the low fluorescence brightness intrinsic to individual fluorescent groups. Such property is generally determined by the fluorescence quantum yield of the fluorescent group. A low fluorescence quantum yield is usually due to energy transfer from the excited electronic state to the vibrational and rotational states of the molecule, a process in which the electronic energy is converted to heat, instead of light. One approach to improve the fluorescence quantum yield of a fluorescent group is to rigidify the dye structure so that the dye has limited vibrational and rotational modes. See, e.g., U.S. Pat. Nos. 5,981,747 and 5,986,093, which describe monomethine cyanine dyes that are rigidified by a two-carbon chain that links the two benzazolium nitrogen atoms. Similarly, in U.S. Pat. No. 6,133,445, trimethine cyanine dyes are rigidified by incorporating the bridge moiety into a three fused ring system. The rigidified cyanine dyes all have significantly improved quantum yields compared to the nonrigidified counterpart dyes. However, the improvements in quantum yield are obtained at the expense of other desirable properties. For example, because of their relatively complex structures, these rigidified dyes typically take several more steps to synthesize than regular cyanine dyes, often with low yields. Highly rigidified dyes may also show a higher tendency to aggregate on proteins. For example, a rigidified cyanine dye has been shown to form dimers even when used at a much lower degree of labeling on proteins than a nonrigidified cyanine (Cooper, et al. Journal of Fluorescence 14, 145(2004)). Furthermore, rigidified trimethine cyanine dyes have shown significantly reduced photostability, compared to regular non-rigidified trimethine cyanine dyes (see, e.g., U.S. Pat. No. 6,133,445).